The overall goal of this work is to develop anatomical, functional, and molecular magnetic resonance imaging (MRI) techniques that allow non-invasive assessment of brain function and apply these tools to study plasticity and learning in the rodent brain. MRI techniques are having a broad impact on understanding brain. Anatomical based MRI has been very useful for separating gray and white matter and detecting numerous brain disorders. Functional MRI techniques enable detection of regions of the brain that are active during a task. Molecular MRI is an emerging area, whose major goal is to image a large variety of processes in tissues. The goal of this project is to translate MRI developments in all these areas to study system level changes that occur in the rodent brain during plasticity and learning. Aim 1: Over the past few years, we have completed studies in the rodent brain that acquired very high temporal and spatial resolution functional MRI (fMRI) to monitor changes in hemodynamics as a surrogate marker of electrical activity during forepaw stimulation. Over the past couple of years we have demonstrated that fMRI from single venuoles can be detected with BOLD fMRI. Recently we have now demonstrated that single arterioles from deep cortex can be effectively imaged using blood volume based MRI techniques. fMRI has long been able to detect individual draining vessels but now we have demonstrated the ability to detect individual vessels in deep tissue. A detailed analysis of spatial and temporal time courses have been done in the different vessel compartments for both blood volume and BOLD based. Consistent with our understanding arteriole blood volume responds earlier that venous BOLD and the two vessel populations can be separated based on time to peak response. In blood volume the arteriole contribution dominates as compared to the tissue or venuole contributions. For BOLD the venuole contribution dominates compared to the arteriole contribution. We will verify our MRI assignment of vessels and determine what fraction of arterioles and venuoles that can be detected. Over the past couple of years we have developed a one dimensional imaging technique that enabled us to achieve 50 micron spatial resolution through the cortex and 50 msec temporal resolution. In somatosensory cortex, fMRI signals start in layer 4 at about 600-800 msec. In motor cortex fMRI onset corresponds to the neural input in mid-cortical areas. In a model where neural input into somatosensory cortex switches to beginning in layer 2/3 and or layer 5 rather than layer 4, the fMRI onset also switches to layer 2/3 and layer 5. This work is consistent with the hypothesis that the onset of fMRI enables extracting information about the onset of neural activity in a brain region. We have begun to asses the determinants of the early hemodynamic response and in particular the role of pericytes. Aim 2: Over the past several years we have demonstrated that manganese chloride enables MRI contrast that defines neural architecture, can monitor activity, and can be used to trace neural connections. Over the last couple of years we have completed the assignment of cortical layers detected using manganese enhanced MRI by comparison to histology and have demonstrated that functional anatomy of several cortical regions of the rodent brain can be defined in individual animals. The ability to detect layers has been applied to a mouse model of neurodegeneration in the olfactory bulb demonstrating the MRI at his level of resolution can detect layer specific degeneration. In addition, we have completed studies that trace laminar inputs of the somatosensory and olfactory pathway that indicate that manganese can indicate changes in synaptic strength. A hippocampal slice preparation has been established to study the synaptic mechanism underlying these MRI properties of manganese. Very high spatial resolution MRI has been obtained and we can detect Mn tracing through the Schaeffer collateral. Finally, in collaboration with Dorian McGavern, we have shown that Mn can enter the brain through the skull at specific locations and once inside the brain trace appropriate neural connections. We are now characterizing the major pathways that Mn can enter the brain via specific pathways through the skull. This opens the very exciting possibility of delivering Mn to specific locations in the brain non-invasively. Aim 3: Over the past few years we established a rodent model that uses peripheral denervation to study brain plasticity in response to the injury. Over the past couple of years we have shown that denervation of the infraorbital nerve leads to large increases in barrel cortex responses along the spared whisker pathway as well as large ipslateral cortical activity consistent with our previous work in the forepaw and hindpaw. fMRI and manganese enhanced MRI predicted a strengthening of thalamo-cortical input along the spared pathway which was verified in slice electrophysiology studies in collaboration with John Isaac's laboratory. Prior to this it was widely believed that the thalamo-cortical input was not capable of strengthening after the critical period. The mechanisms of this strengthening are under study. Over the past year we have conclusively shown that the denervation is causing a re-activation of the ability of this synapse to demonstrate long term potentiation. This LTP is occurring via activation of silent synapses. Measurements of AMPA/NMDA ratios and failure rates after minimal stimulation indicate that there is an opening of silent synapses underlying LTP. While LTP is not dependent on the NR2B subunit, results injecting NR2B inhibitors into the rodent cortex indicate that NR2B is important for establishing silent synapses. Evidence from manganese based track tracing shows a strengthening of input into layer 2/3 and 5 from the contralateral cortex to the ipsilateral cortex from the healthy whiskers. We have demonstrated that we can use optogenetic stimulation to study this input and have shown that the mouse has similar plasticity to the rat. Over the next year we will use slice electrophysiology to verify this strengthening in analogy to the work done on the thalamo-cortical synapse and study the mechanisms for the synaptic changes. To the best of our knowledge this work represents the first time MRI has guided the identification of specific synaptic changes. Aim 4: Progress in this aim over the past year has been slow due to lack of a specific person to carry out the work. However, Over the past few years we have begun to explore the use of advanced MRI tools for studying simple learning paradigms in the rodent. In order to accomplish this we have been developing techniques that will enable routine fMRI in awake rodents. While fMRI is widely performed in humans and awake primates there have only been a few scattered studies on awake rodents. Training regimens and techniques to hold the head have been established. This is important to lay the ground work for the best types of stimuli that give good fMRI responses in the awake rodent to better design behavioral procedures that are consistent with fMRI. In the coming years we will begin to see if fMRI can detect changes in circuit level activity during fear conditioning in the rodent brain.